Known methods for ‘next generation’ (e.g., polony) sequencing use degenerate oligonucleotides and protein ligases to discriminate among oligonucleotides with perfect match at one or more positions relative to mismatched oligonucleotides (See, e.g., Shendure et al. (2005) Science 309:1728; Applied Biosystems Solid; and Complete Genomics). Once ligated to an anchor oligonucleotide bound to an immobilized template, the solid phase is extensively washed to remove free fluor-oligonucleotides and then visualized with a digital camera. The oligonucleotides are typically labeled with fluorophores such that a given emission wavelength corresponds with given base(s) (e.g., A, C, G, T) at specific locations in the oligonucleotide bound to the template (e.g., unknown) DNA.
Methods employing degenerate oligonucleotides and protein ligases suffer from many disadvantages. In one approach, the coupling to each monomer goes to 50% completion in 0.15-2.5 hours and is, thus, too slow to be useful, perhaps in part because there is too little hybridization energy from a single base-pair (Griesang et al. (2006) Angew. Chem. Int. Ed. Engl. 45:6144). Another disadvantage of these art-known methods is that photocleavable protection chemistry requires intense irradiation with UV light, which can damage the DNA template to be sequenced after just a few deprotections.